Evaluation of the mutant fraction using standard mutagenesis assays can be inaccurate because mutant and wild-type cells are usually mixed during the expression phase. If some mutant progenitors or mutants grow more slowly than the wild-type cells during the expression period, there will be a decrease in the mutant wild-type cells during the expression period, there will be a decrease in the mutant to wild-type ratio and the mutant fraction will not accurately represent the number of mutational events that occurred. In order to accurately quantitate the mutagenic response of the cells, we have developed an in situ procedure which segregates and immobilizes cells during expression. In these experiments, we plated L5178Y tk+/- mouse cells in semisolid medium immediately after treatment. The cells grew and formed microcolonies. We added the selective agent, TFT, as an overlay at specified times, permitting only TFTr cells to survive. In this procedure, each mutation is captured as an individual colony: consequently, the measured mutation fraction accurately reflects the mutational events that occur at the selected locus. In addition, the induced mutant colonies arising in the agar are the result of independent mutational events. We have developed the in situ assay protocol for the L5178Y cell line and showed that the spontaneous mutation rate measured in the in situ assay was 50-fold greater than when the cells expressed the phenotype in suspension. We evaluated the effect of chemical treatment on the mutation rate as a function of the time to TFT addition and generated expression curves for 6 compounds. The numbers of TFTr colonies produced at various times after treatment indicated that chemically-treated cultures had higher mutation fractions than the solvent controls. These results document the feasibility of quantitating induced mutation rates using the in situ protocol. This approach has also been applied to AS52 cell mutation assays at the gpt gene. We believe that this method should be considered for routine evaluation of the potential mutagenic effects of chemicals in mammalian cells in vitro.